Aromatic aldehydes with sustained and enhanced in vitro and in vivo pharmacologic activity to treat sickle cell disease

ABSTRACT

Compounds and methods for preventing and/or treating one or more symptoms of sickle cell diseases (SCD) by administering at least one of the compounds are provided. The compounds are based on vanillin which is chemically modified to increase bioavailability and activity, e.g. so that the compounds bind to the F helix of hemoglobin (Hb) and prevent adhesion of red blood cells (RBCs).

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under contract numberMD009124 awarded by the National Institutes of Health. The United Statesgovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The invention generally relates to compounds that exhibit enhancedactivity and stability for the treatment of sickle cell disease (SCD).In particular, the compounds are vanillin-based aromatic aldehydes inwhich the alcohol moiety is converted into aryl or alkyl amides andesters or inorganic phosphate esters, and in which the aldehydefunctional (CHO) group is optionally replaced by a protected aldehydemoiety (promoiety).

Description of Related Art

Hemoglobin (Hb) functions to oxygenate tissue by equilibrating betweentwo allosteric states: a tense (T) state, which exhibits low-affinityfor ligand, and a relaxed (R) state, which exhibits high affinity forligand.¹ Sickle cell disease (SCD) is an inherited hematologic disorderand occurs as a result of replacement of βGlu6 with βVal6 in Hb, formingsickle Hb (Hb S).² Under hypoxia or low oxygen (O₂) tension, which leadsto an increased concentration of the low-affinity deoxygenated (T-state)Hb, Hb S polymerizes into long, rigid, and insoluble fibers resulting insickling of red blood cells (RBCs). The polymer which is initiated bythe primary interaction involving βVal6 is stabilized by several othersecondary contacts between the Hb S molecules.³ Hypoxia-induced sicklingleads to several secondary pathophysiological events, e.g. adhesion ofRBCs to tissue endothelium, oxidative stress/damage, hemolysis (rupture)of RBCs, inflammation, vaso-occlusion, impaired microvascular bloodflow, decreased vascular nitric oxide bioavailability, painful crises,morbidity and mortality.⁴ The only drugs currently approved for treatingSCD are hydroxyurea (HU) and more recently, Endari™ (L-glutamine oralpowder). HU induces γ-globin expression to form fetal Hb (HbF).⁵However, not all patients respond to HU and/or Endari™, and HU can causemyelosuppression, a life-threatening side effect. Furthermore, the drugremains inaccessible in many parts of the world. The need for alternatemodes of therapy remains extremely pressing, especially in the face ofthe significant mortality, morbidity, healthcare disparities and publichealth burden imposed by SCD.

Vanillin (FIG. 1) and several of its analogs and derivatives havepreviously been studied for their antisickling activity.^(1,6,7)Specifically, the compounds form Schiff-base adduct with Hb to stabilizethe high-affinity oxygenated R-state Hb (in the R2 conformation)relative to the low-affinity deoxygenated T-state Hb, resulting inincrease in Hb affinity for oxygen. By increasing Hb affinity foroxygen, the compounds prevent the hypoxia-induced primarypathophysiology of Hb S polymerization and RBC sickling, andconcomitantly ameliorate several of the cascading secondary adverseevents. Schiff-base interaction between the compound's aldehyde groupand the N-terminal valine amino group of α globin chains (Val1) existsin equilibrium between bound adduct complex and unbound Hb and freecompound. For a stable Schiff-base complex and consequently an effectivepharmacologic outcome, these compounds should make strong interactionswith Hb, and exhibit a slow rate of dissociation. Vanillin does not formstrong interactions with the protein, and consequently its dissociationfrom the protein is fast, consistent with vanillin's sub-optimalbiological activity. In addition to vanillin's weak interactions withthe protein, it also undergoes significant metabolism of the aldehyde,which is the active component of the molecule, resulting in poorpharmacokinetic properties, including lack of bioavailability.

The crystal structures of vanillin and several of its analogs andderivatives have been elucidated to explain how binding of thesemolecules leads to stabilization of the R-state Hb and consequentlyreduces or prevents hypoxia-induced Hb S polymerization and RBCsickling.^(1,8) Based on these previous studies, a new generation ofcompounds (termed TD) were developed by coupling a hydroxylmethylpyridinyl to the benzaldehyde of the vanillin (FIG. 1). Although the TDcompounds showed a significantly more potent effect than vanillin, likevanillin, these compounds also undergo significant metabolism leading tosub-optimal pharmacokinetic properties, e.g. short duration ofpharmacologic action and low bioavailability. These properties wouldnecessitate the use of frequent and very high doses of such compounds,which is undesirable for treatment of a chronic disease, and hence theirdevelopment has not been pursued.

There is a pressing need to develop new anti-sickling agents that bindwith higher affinity to Hb and do not undergo significant metabolism,that exhibit a long duration of pharmacologic action and have a highlevel of bioavailability.

SUMMARY OF THE INVENTION

Other features and advantages of the present invention will be set forthin the description of invention that follows, and in part will beapparent from the description or may be learned by practice of theinvention. The invention will be realized and attained by thecompositions and methods particularly pointed out in the writtendescription and claims hereof.

Provided herein are new, potent anti-sickling agents that have sustainedand enhanced antisickling pharmacologic activity in vitro and in vivo,compared to prior art compounds. Activity is sustained due to resistanceto in vivo metabolism and activity is increased due to a dualantisickling mechanism of action: i) the compounds increase Hb affinityfor oxygen and ii) directly destabilize polymers formed by sickled RBCs.These are critical properties for a drug that is repeatedly administeredto treat a chronic condition such as sickle cell anemia.

It is an object of this invention to a vanillin-derived compound havingFormula I:

where R1 and R2 are the same or different and are H; hydroxyl; halogen;or a substituted or unsubstituted: alkyl, alkoxy, aryl, O-aryl,cycloalkane or heterocycle; R3 is alkyl ester, aryl ester, alkylamide,arylamide, phosphate or sulfate; M and Q are the same or different andare O or (CH₂)n where n=0-6; X, Y, Z, W and V are the same or differentand are independently H, C, N, S or O; m=0-6, and P=CHO or a promoiety;or a pharmaceutically acceptable salt thereof. In some aspects, thevanillin-derived compound is designed to bind to the F helix ofhemoglobin (Hb). In additional aspects, P is CHO. In further aspects, Pis a protected aldehyde group or promoiety. In other aspects, thepromoiety is

where R4 is H or a linear or branched C1-C5 alkyl; and where the bondmarked with * bonds directly to a carbon of the benzene ring. In yetfurther aspects, the vanillin-derived compound is selected from thegroup consisting of:

In additional aspects, the vanillin-derived compound is

In yet further aspect, the vanillin-derived compound is

In some aspects, the vanillin-derived compound is a HCl salt.

Also provided is a composition comprising at least one vanillin-derivedcompound as disclosed herein. In some aspects, the composition is in aform for oral administration.

Also provided is a method of preventing or treating one or more symptomsor conditions of sickle cell disease (SCD) in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of at least one vanillin-derived compound as disclosed herein. Insome aspects, the one or more symptoms or conditions of SCD are selectedfrom the group consisting of HbS polymerization, red blood cell (RBC)sickling, adhesion of RBCs to tissue endothelium, oxidative stressand/or damage, hemolysis of RBCs, inflammation, vaso-occlusion, impairedmicrovascular blood flow, a decrease in vascular nitric oxidebioavailability, pain, and death. In some aspects, the step ofadministering is performed orally.

Also provided is a method of preventing or treating one or more symptomsof hypoxia in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of at least onevanillin-derived compound disclosed herein. In some aspects, the step ofadministering is performed orally.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Structures of vanillin and a prototype TD compound, TD-7.

FIG. 2: Structures of exemplary “PP” compounds.

FIGS. 3A and 3B. A, crystal structure of R2-state Hb in complex withPP9; B, F helix interactions.

FIG. 4. Dose-dependent in vitro oxygen equilibrium curve (OEC) of PPcompounds with normal whole blood.

FIGS. 5A and 5B. Time-dependent in vitro Hb modification by PP compoundswith normal whole blood. A, PP compounds 2, 3, 4, 8, 9, and 12; B, PPcompounds 6, 10, 7, 11, 14 and 13.

FIG. 6. Dose-dependent in vitro sickling inhibition by PP compounds withsickle red blood cells.

FIG. 7. Dose-dependent in vitro OEC of PP compounds with sickle redblood cells.

FIG. 8. Dose-dependent in vitro Hb modification by PP compounds withsickle red blood cells.

FIG. 9. Antisickling effects of PP compounds and other aromaticaldehydes under 2.5% oxygen gas vs 100% nitrogen gas.

FIGS. 10A and 10B. In Vivo Pharmacologic effect of PP compounds inwild-type mice. A, time-dependent Hb modification; B, time-dependent Hboxygen affinity shift.

FIGS. 11A and 11B. In Vivo Pharmacologic effect of PP14 in wild-typemice using optimized vehicle for drug administration. A, time dependentmodification of intracellular Hb in wild-type mice after oral (n=3), orIP (n=2) administration of 150 mg/kg PP14; B, time-dependent (5 hrs) Hboxygen affinity shift in wild-type mice after oral (n=3), or IP (n=2)administration of 150 mg/kg PP14.

DETAILED DESCRIPTION

Disclosed herein are vanillin-based compounds in which an alcohol moietyon the pyridine ring is modified into aryl or alkyl amides and esters orinorganic phosphate esters. This derivatization renders the alcoholresistant to in vivo metabolism, leading to more sustained and enhancedpharmacologic activities. In addition, the bulky ester or amide moietyof these novel compounds makes novel interactions with the F helix ofHb. As a result, these compounds exhibit a dual antisickling mechanismof action: 1) the compounds increase the affinity of HbS for oxygen,making less HbS available for sickling; and 2) the compounds interferedirectly with HbS polymer formation. In addition, the CHO group ofvanillin is optionally replaced by a protected aldehyde group(promoiety, pharmacophore), thereby forming e.g. a prodrug to furtherincrease bioavailability. A pharmacophore is a part of a molecularstructure or compound that is responsible for a particular biological orpharmacological interaction.

As noted in the Background section above, hypoxia or low oxygen (O₂)tension leads to an increased concentration of low-affinity deoxygenated(T-state) Hb. The compounds disclosed herein bind to liganded HbS andhold the target protein in a high-affinity oxygenated (relaxed) statethereby decreasing the concentration of deoxygenated HbS, as well aspreventing premature and fast release of the bound oxygen prior toliganded Hb reaching tissue beds.

Furthermore, deoxygenated HbS typically polymerize into long, rigid, andinsoluble fibers, resulting in sickling of red blood cells (RBCs). The Fhelix of HbS is very important in stabilizing these polymers throughsecondary interactions with adjacent HbS. Without being bound by theory,it is believed that the bulky ester or amide moieties, or the chargedsulphate or phosphate groups of the compounds interact with the surfacelocated F helix of Hb, and this interaction leads to stereospecificinhibition of polymer formation by HbS. In other words, binding of theseligands to the F helix induces a conformation change, occludes the Fhelix, and abrogates interactions between HbS, thereby weakening thepolymer and preventing sickling.

The Compounds

Compounds disclosed herein are based on or derived from vanillin. Thecompounds, or a functional group or substituent of the compounds (e.g.the ester, amide, sulphate or phosphate groups) binds to the F helix ofhemoglobin (Hb) and prevents or decreases the interaction between HbSmolecules and thus prevent sickling of red blood cells (RBCs).

The compounds have a generic Formula I:

wherein:

-   -   R1 and R2 are the same or different and are H; hydroxyl;        halogen; or substituted or unsubstituted: alkyl, alkoxy, aryl,        O-aryl, cycloalkyl or heterocyclic;    -   R3 is an alkyl ester, aryl ester, an alkylamide, an arylamide,        phosphate or sulfate;    -   M and Q are the same or different and are O or (CH₂)n where        n=0-6 (e.g. 0, 1, 2, 3, 4, 5, or 6);    -   X, Y, Z, W and V are the same or different and are        (independently) H, C, N, S or O;    -   m=0-6 (e.g. 0, 1, 2, 3, 4, 5, or 6), and    -   P=CHO or a promoiety.

Pharmaceutically acceptable salts of the compounds are also encompassed.

Examples of suitable promoieties include but are not limited to thethiazolidine:

where R4 is H or a linear or branched C1-C5 alkyl, and where the bondmarked with * bonds directly to a carbon of the benzene ring. Othermeans of protecting the aldehyde, include but not limited to conversionof the aldehyde to the corresponding imine, acetal, hemiacetal, ester,or alcohol.

Exemplary halogens include but are not limited to: Cl, Br, I and F.

Exemplary alkyl groups include but are not limited to: linear orbranched C1-C12 alkyl e.g. methyl, ethyl, propyl, isopropyl, butyl (e.g.n-butyl, secondary butyl, isobutyl, tertiary butyl), pentyl (e.g.n-pentyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3-pentyl),hexyl, etc.) each of which may be substituted or unsubstituted. Thealkyl group may be optionally substituted with one, two, or threesubstituents, or, in the case of alkyl groups of two carbons or more,four substituents e.g. alkoxy, alkylsulfayl, amino, azido, halo,heterocyclyle)oxy, hydroxyl, nitro, oxo, thioalkoxy, thiol, etc.

Alkoxy refers to an alkyl group singularly bonded to oxygen (R—O), i.e.an alkyl group having an oxygen radical attached thereto. In someaspects, the R group of the alkoxy is a (C₁-C₁₂) alkyl (see above forexemplary alkyls) having an oxygen radical attached thereto. Exemplaryalkoxy groups include but are not limited to: methoxy, ethoxy, propoxy(e.g., n-propoxy and isopropoxy), t-butoxy, and the like, each of whichmay be substituted or unsubstituted. In some embodiments, the alkylgroup or cyclic alkyl ring can be further substituted with 1, 2, 3, or 4substituent groups as defined herein (e.g., O, hydroxyl, alkoxy, etc.).

As used herein, “aryl” refers to any functional group or substituentthat is or is derived from an aromatic ring, e.g. an aryl is a radicalderived from an aromatic hydrocarbon by removal of a hydrogen atom. Thearomatic hydrocarbon may be heteroaromatic (one or more atoms in thering is/are not carbon and are instead e.g. O, N, S, etc.); polycyclic(containing two or more aromatic rings); or substituted (aromatic ringshaving other functional groups attached, e.g. alkyl, alkenyl, phenyl,aldehyde, hydroxyl, sulfhydryl, carboxyl, carbonyl, amide, amine,nitrate, sulfate, phosphate, pyridyl, etc.). Examples of aryl groupsinclude but are not limited to: an aromatic hydrocarbon such as phenyl,naphthyl, thienyl, indolyl, tolyl, furyl, pyridyl, anthracenyl,fluorenyl, indanyl, indenyl, etc. each of which may be heteroaromaticand/or substituted or unsubstituted.

“O-aryl” refers to an oxygen atom bonded to an aryl group, as describedabove, i.e. a radical derived from an aromatic hydrocarbon by removal ofa hydrogen atom. O-aryl groups which are used in the compounds describedherein include but are not limited to those listed above for aryl, eachof which may be substituted or unsubstituted.

A cycloalkane is a cyclic compound in which all atoms of the ring (orrings) are C and the bonds between C atoms are saturated. As usedherein, cycloalkyl refers to the radical (form that has an unpairedvalence electron) and can form a bond with another entity. Cycloalkylcompounds may be unicyclic or polycyclic (e.g. bicyclic, tricyclic,etc.); and polycyclic substituents comprising more (e.g. 4, 5, 6, etc.)rings are also encompassed. If the system is polycyclic, the rings inthe systems may have the same number of atoms (e.g. a bicyclic ringcomprising two 6-membered rings); or the rings of the system may havetwo different types of rings (e.g. a bicyclic ring comprising one6-membered ring and one 5-membered ring), and may be e.g. fused orbridged. Examples of cycloalkyls include but are not limited to:cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, butyl, cyclopentyl, hexyl, heptyl, octyl, etc., andpolycyclic combinations of these such as: decalin, camphorquinone,polyquinane, and the like. The cycloalkyl may be substituted orunsubstituted.

A heterocyclic compound or ring structure is a cyclic compound (e.g. acycloalkane or aryl compound) that has atoms of at least two differentelements as members of its ring(s). Generally, each ring of the systemhas about 4, 5, 6, or 7 atoms, at least one of which is carbon, and atleast one of which is not carbon. For example, 1 or 2 atoms may benon-carbon atoms while the rest are carbon atoms. The rings may or maynot contain double bonds, e.g. one or more C═C bonds. The heteroatomsare, for example, N, O, P or S, but other heteroatoms are not excluded.Exemplary heterocyclic systems are unicyclic, bicyclic or tricyclic;however, systems comprising more rings are also encompassed. If thesystem is multicyclic, the rings in the systems may have the same numberof atoms (e.g. a bicyclic ring comprising two 6-membered rings) and thetwo rings may be the same or different; or the rings of the system mayhave two different types of rings (e.g. a bicyclic ring comprising one6-membered ring and one 5-membered ring). Examples of heterocyclicsystems that may be present in the compounds disclosed herein includebut are not limited to: oxazole, pyrazoline, imidazole, pyrazole,pyrazine, purine, indoline, quinolone, pteridine, indene, piperidine,tetrahydrofuran, pyridine, pyrimidine, thiophene, pyrrole, furan,quinoline, benzothiophene, indole, benzofuran, heterocycles comprisingtwo substituted benzene rings such as acridine, dibenzothiophene,carbazole, dibenzofuran, etc.

Alkyl esters and aryl esters are esters (chemical compounds derived froman organic or inorganic acid in which at least one —OH (hydroxyl) groupis replaced by an —O-alkyl (alkoxy) group or an —O-aryl group, with thealkyl and aryl portions being those defined above.

Alkylamides and arylamide are amides (acid derivatives with the generalformula R′—CO—NH₂, where R′ is an alkyl or aryl group as describedabove.

Pharmaceutically acceptable salts of the compounds are also encompassed.Examples of such salts include, but are not limited to, salts formedwith inorganic acids (for example, hydrochloric acid (HCl), hydrobromicacid, sulfuric acid, phosphoric acid, nitric acid, and the like), andsalts formed with organic acids such as acetic acid, oxalic acid,tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid,tannic acid, palmoic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, naphthalenedisulfonic acid, methanesulfonicacid, p-toluenesulfonic acid and polygalacturonic acid. Other saltsinclude pharmaceutically acceptable quaternary salts known by thoseskilled in the art, which specifically include the quaternary ammoniumsalt of the formula —NR+Z—, wherein R is hydrogen, alkyl, or benzyl, andZ is a counterion, including chloride, bromide, iodide, —O-alkyl,toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate(such as benzoate, succinate, acetate, glycolate, maleate, malate,citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate,benzyloate, and diphenylacetate).

Exemplary Compounds

Exemplary compounds of the invention include but are not limited to:

Additional exemplary compounds include but are not limited to:

Mechanistically, these compounds increase the hemoglobin oxygen affinityand concomitantly exhibit antisickling activity in part via aSchiff-base interaction between the aldehyde moiety and the N-terminalαVal1 nitrogen of relaxed state hemoglobin. Like all aldehydes, thealdehyde moiety of the compounds is susceptible to rapid and significantmetabolism, e.g. by aldehyde dehydrogenase, into the inactivecarboxylate derivative that could potentially shorten the compoundspharmacologic effect. Consistently, several aromatic aldehydeantisickling agents, e.g. 5-HMF have failed in human clinical studiesbecause of such metabolic shortcomings. To overcome such potentialdisruptive metabolism, in some aspect, the aldehyde group of thecompounds presented herein is protected, e.g. via a coupling reactionwith L-cysteine to form the thiazolidine complex (Zhang et al. Br JHaematol. 2004; 125:788-795). The promoiety or prodrug compounds exhibitimproved bioavailability and/or a longer half-life due to decreasedaldehyde metabolism. This permits the drugs to be administered i) atlower doses and/or ii) less frequently, while still maintaining thebeneficial therapeutic effects of the unprotected aldehyde, therebyminimizing side effects and/or increasing patient compliance withadministration.

Thus, in some aspects, the compounds described and depicted above may befurther rendered resistant to in vivo metabolism by replacing the CHOfunctional group with a protecting group. Examples of suitablepromoieties (pharmacophores) include but are not limited to

where R4 is H or a linear or branched C1-C5 alkyl; and where the bondmarked with * bonds directly to a carbon of the benzene ring. An exampleof this promoiety is that which is formed when L-cysteine forms athiazolidine complex:

where * indicates the point of attachment to C of the benzene ring.Other routes to form the thiazolidine, include but not limited to theuse of cysteamine and amino thiol.

Other means of protecting the aldehyde include but are not limited toconversion of the aldehyde to the corresponding imine, acetal,hemiacetal, ester, or alcohol.

Examples of compounds that comprise a promoiety include but are notlimited to:

A promoiety may be included in or on any of the compounds disclosedherein.

Advantages of the Compounds

The compounds described herein exhibit improved pharmacologic activity,i.e. more potency and/or increased (lengthened, longer-lasting, etc.)half-lives, and/or improved bioavailability under physiologicalconditions (e.g. in circulation, in plasma, etc.) compared to othervanillin-based compounds. For example, the compounds exhibit in vivohalf-lives of at least about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5,5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5or 12.0 hours, or even longer, e.g. about 12 to 36 hours, i.e. about 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 hours. In some aspects,compared to vanillin or TD, the compounds exhibit 1, 2, 3, 4, 5, 6, 7,8, 9, or 10-fold or more greater potency in terms of preventing the HbSsickling and/or the formation of sickled HbS polymers. Thus, in someaspects, the compositions comprising the compounds are administered lessfrequently and/or at lower doses than would be required for prior artcompounds, yet the same or an increased level of beneficial effects isexperienced by subjects receiving the compounds.

Exemplary Methods of Treatment Using the Prodrugs or Derivatives

The compounds described herein are used to treat or prophylacticallytreat diseases and/or conditions related to hypoxia, such as SCD andother diseases/conditions described below. As used herein,“prophylactically treat” (“prophylactic treatment”, “prophylacticallytreating” etc.) and “prevent” (“prevention”, “preventing” etc.) refer towarding off or averting the occurrence of at least one symptom of adisease or unwanted condition (such as at least one symptom of SCD), byprophylactic administration of a composition comprising at least onecompound as described herein, to a subject in need thereof. Generally,“prophylactic” or “prophylaxis” relates to a reduction in the likelihoodof the patient developing a disorder or a symptom of a disorder.Typically, the subject is considered by one of skill in the art to be atrisk of or susceptible to developing at least one symptom of the diseaseor unwanted condition, or is considered to be likely to develop at leastone symptom of the disease/condition in the absence of medicalintervention. In some aspects, for “prevention” or “prophylactictreatment”, administration occurs before the subject has, or is known orconfirmed to have, symptoms of the disease (condition, disorder,syndrome, etc.; unless otherwise indicated, these terms are usedinterchangeably herein). In other words, symptoms may not yet be overtor observable, or may be very “early stage” symptoms. The subject may beconsidered at risk due to a variety of factors, including but notlimited to: genetic predisposition; evidence of “early” symptoms; testssuch as blood tests, etc. In such aspects, treatment of the subject mayprevent the noxious or harmful effects or outcomes (results) of fullblown disease. “Prevention” or “prophylactic treatment” of a disease orcondition may involve completely preventing the occurrence of detectablesymptoms, or, alternatively, may involve lessening or attenuating thedegree, severity or duration of at least one symptom of the disease thatwould otherwise occur in the absence of the medical interventionsprovided herein.

“Treat” (treatment, treating, etc.) as used herein refers toadministering at least one composition comprising a compound asdescribed herein, to a subject that already exhibits at least onesymptom of a disease such as SCD. In other words, at least one parameterthat is known to be associated with the disease has been measured,detected, experienced or observed in the subject. For example, thesymptom may be the primary pathophysiology of hypoxia-induced RBCsickling associated with sickle cell disease. In addition, the compoundsdisclosed herein ameliorate several of the cascading secondary adverseevents of SCD, including adhesion of RBCs to tissue endothelium,oxidative stress, hemolysis of RBCs, decreased vascular NObioavailability, vaso-occlusion, impaired microvascular blood flow,increased blood pressure, and painful crises, e.g. due to polymerizationof RBCs. For example, the compounds generally do one or more of thefollowing: increase O₂-affinity of HbS; decrease fiber formation; reducesickle cell mechanical fragility; reduce RBC hemolysis; attenuatehypoxia-induced cell necrosis and apoptosis; improve microvascularfunction (e.g. during recovery from hemorrhagic shock); results inhemodynamic and oxygenation benefits during hypoxia (e.g. maintenance ofblood pressure and heart rate; preservation of microvascular blood flow;reduction in heart and brain hypoxia areas); reduce pain; decreaselactate dehydrogenase and/or RBC hemolysis; reduce diastolic bloodpressure; increase blood oxygen levels (S_(p)O₂) during hypoxiachallenge; etc.

“Treatment” of a disease involves the lessening or attenuation, or insome instances, the complete eradication, of at least one symptom of thedisease that was present prior to or at the time of administration ofthe composition.

Exemplary Compositions and Methods of Administration

Provided herein are compositions comprising at least one compound asdescribed herein, and methods of administering the same to treat e.g.SCD, hypoxia, etc. Implementation of the methods generally involvesidentifying patients suffering from or at risk of developing a diseaseor condition described herein (for example SCD or hypoxia), andadministering a composition as described herein by an appropriate route.The exact dosage to be administered may vary depending on the age,gender, weight and overall health status of the individual patient,severity of disease symptoms, or on other treatments being received bythe patient, as well as the extent or progression of the diseasecondition being treated and the precise etiology of the disease.However, in general for administration to mammals (e.g. humans),sufficient composition is administered to achieve dosages in the rangeof from about 0.1 to about 1000 mg or more per kg of body weight per 24hr., e.g. from about 1 to about 500 mg, 5 to 100, or 10-50 mg per kg ofbody weight per 24 hr. Generally, a therapeutically effective dose isfrom about 50 to about 150 mg per kg of body weight per 24 hr. The dosewill vary with the route of administration, the bioavailability, and theparticular formulation that is administered, as well as according to thenature of the malady that is being prevented or treated.

The compositions are generally administered in a pharmaceuticallyacceptable formulation which includes suitable excipients, elixirs,binders, and the like (generally referred to as “pharmaceutically andphysiologically acceptable carriers”), which are pharmaceuticallyacceptable and compatible with the active ingredients. The prodrugs orderivatives may be present in the formulation as pharmaceuticallyacceptable salts (e.g. alkali metal salts such as sodium, potassium,calcium or lithium salts, ammonium, etc.) or as other complexes. Itshould be understood that the pharmaceutically acceptable formulationsinclude solid, semi-solid, and liquid materials conventionally utilizedto prepare solid, semi-solid and liquid dosage forms such as tablets,capsules, liquids, aerosolized dosage forms, and various injectableforms (e.g. forms for intravenous administration), etc. Suitablepharmaceutical carriers include but are not limited to inert soliddiluents or fillers, sterile aqueous solutions and various organicsolvents. Examples of solid carriers (diluents, excipients) includelactose, starch, conventional disintegrating agents, coatings, lactose,terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesiumstearate, stearic acid and lower alkyl ethers of cellulose. Examples ofliquid carriers include but are not limited to various aqueous or oilbased vehicles, saline, dextrose, glycerol, ethanol, isopropanol,phosphate buffer, syrup, peanut oil, olive oil, phospholipids, fattyacids, fatty acid amines, polyoxyethylene, isopropyl myristate, ethylcocoate, octyl cocoate, polyoxyethylenated hydrogenated castor oil,paraffin, liquid paraffin, propylene glycol, celluloses, parabens,stearyl alcohol, polyethylene glycol, isopropyl myristate,phenoxyethanol, and the like, or combinations thereof. Water may be usedas the carrier for the preparation of compositions which may alsoinclude conventional buffers and agents to render the compositionisotonic. Oral dosage forms may include various thickeners, flavorings,diluents, emulsifiers, dispersing aids, binders, coatings and the like.The composition of the present disclosure may contain any suchadditional ingredients so as to provide the composition in a formsuitable for the intended route of administration. In addition, thecomposition may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, and the like.Similarly, the carrier or diluent may include any sustained releasematerial known in the art, such as glycerol monostearate or glyceroldistearate, alone or mixed with wax. Other potential additives and othermaterials (preferably those which are generally regarded as safe [GRAS])include: colorants; flavorings; surfactants (TWEEN®, oleic acid, etc.);and solvents, stabilizers, binders or encapsulants (lactose, liposomes,etc.). Preservatives such as methyl paraben or benzalkium chloride mayalso be used. Depending on the formulation, it is expected that theactive components (e.g. at least one prodrug or derivative) will bepresent at about 1% to about 99% of the composition and the vehicular“carrier” will constitute about 1% to about 99% of the composition. Thepharmaceutical compositions of the present disclosure may include anysuitable pharmaceutically acceptable additives or adjuncts to the extentthat they do not hinder or interfere with the therapeutic effect(s) ofthe composition. Still other suitable formulations for use in thepresent disclosure can be found, for example in Remington'sPharmaceutical Sciences 22nd edition, Allen, Loyd V., Jr editor(September 2012); and Akers, Michael J. Sterile Drug Products:Formulation, Packaging, Manufacturing and Quality; publisher InformaHealthcare (2010).

The compositions (preparations) of the present disclosure are formulatedfor administration by any of the many suitable means which are known tothose of skill in the art, including but not limited to: orally, byinjection, rectally, by inhalation, intravaginally, intranasally,topically, as eye drops, via sprays, transdermally, sublingually, byrectal and buccal delivery, by inhalation of an aerosol, by microneedledelivery, etc. In some aspects, the mode of administration is oral, byinjection or intravenously, preferably via an orally administered pill.

The administration of the compound of the present disclosure may beintermittent, or at a gradual or continuous, constant or controlled rate(e.g. in a sustained release formulation which further extends the timeof bioavailability). In addition, the time of day and the number oftimes per day that the pharmaceutical formulation is administered varyand are best determined by a skilled practitioner such as a physician.Generally, the compounds are administered at least once a day, and maybe administered e.g. 2, 3, 4, or more times per day. During a crisis,administration may be more frequent, e.g. continuously via IV.

Administration of the compound by any means may be carried out as asingle mode of therapy, or in conjunction with other therapies andtreatment modalities, e.g. antibiotics, pain medication, hydroxyurea,vaccinations, blood transfusions, provision of supplemental oxygen, genetherapy, nitric oxide, drugs to boost fetal hemoglobin production,statins, vanillin, TD compounds, etc. In addition, if hypoxia due to aheart condition is the indication, then additional treatments for heartdisease may be provided, including surgery. Other treatment optionsinclude various neutraceuticals, diet regimens, exercise, etc. “Inconjunction with” refers to both administration of a separatepreparation of the one or more additional agents, and to inclusion ofthe one or more additional agents in a composition of the presentdisclosure.

The subject to whom the composition is administered is generally amammal, frequently a human, but this is not always the case. Veterinaryapplications of this technology are also contemplated, e.g. forcompanion pets (cats, dogs, etc.), or for livestock and farm animals,for horses, and even for “wild” animals that have special value or thatare under the care of a veterinarian, e.g. animals in preserves or zoos,injured animals that are being rehabilitated, etc.

Diseases and Conditions That Are Treated

In some aspects, the disease or condition that is prevented or treatedas described herein is sickle cell disease and pathophysiologiesassociated with SCD, such as hypoxia-induced RBC sickling. In addition,the compounds ameliorate cascading secondary adverse events, includingadhesion of RBCs to tissue endothelium, oxidative stress, hemolysis ofRBCs, decreased vascular NO bioavailability, vaso-occlusion, impairedmicrovascular blood flow, increased blood pressure, and painful crises.In addition, the compounds increase O₂-affinity of HbS, decrease fiberformation, reduce sickle cell mechanical fragility, increase bloodoxygen levels (SpO₂) and reduce RBC hemolysis.

In other aspects, the compounds are used to treat or prevent symptoms ofhypoxia that is or is not related to SCD. As used herein hypoxia (alsoknown as hypoxiation) is a condition in which the body or a region ofthe body is deprived of adequate oxygen supply at the tissue level.Hypoxia is classified as either generalized, affecting the whole body,or local, affecting a region of the body. There are four types ofhypoxia: (1) the hypoxemic type, in which the oxygen pressure in theblood going to the tissues is too low to saturate the hemoglobin; (2)the anemic type, in which the amount of functional hemoglobin is toosmall, and hence the capacity of the blood to carry oxygen is too low;(3) the stagnant type, in which the blood is or may be normal but theflow of blood to the tissues is reduced or unevenly distributed; and (4)the histotoxic type, in which the tissue and/or cells are poisoned andare therefore unable to make proper use of oxygen. Diseases of theblood, the heart and circulation, and the lungs may all produce someform of hypoxia.

Generalized hypoxia occurs, for example, in healthy people when theyascend to high altitude, where it causes altitude sickness leading topotentially fatal complications such as high altitude pulmonary edema(HAPE) and high altitude cerebral edema (HACE). Hypoxia also occurs inhealthy individuals when breathing mixtures of gases with a low oxygencontent, e.g. while diving underwater or when in outerspace, andespecially when using closed-circuit rebreather systems that control theamount of oxygen in the supplied air. Hypoxia also occurs as aconsequence of preterm birth in the neonate due to immature lungdevelopment. Hypoxia resulting from ischemia (insufficient blood flow toa tissue or organ), is referred to as ‘ischemic hypoxia’ and is causedby e.g. an embolic event, a heart attack that decreases overall bloodflow, or trauma to a tissue that results in damage, or may bepurposefully induced in some medical procedures, e.g. implantation of astent, application of a tourniquet, etc. Diseases such as peripheralvascular disease can cause local hypoxia. Other causes includealterations in respiratory drive, such as in respiratory alkalosis,physiological or pathological shunting of blood, diseases interfering inlung function resulting in a ventilation-perfusion mismatch, such as apulmonary embolus, or alterations in the partial pressure of oxygen inthe environment or lung alveoli. When hemoglobin is deficient, anemiacan result and can cause ‘anaemic hypoxia’ if tissue perfusion isdecreased. Carbon monoxide poisoning can cause hypoxia, either acutely,as with smoke intoxication, or over a period of time, as with cigarettesmoking or exposure to smog. Certain odorless asphyxiant gases (e.g.nitrogen, methane, etc.) induce hypoxia as does cyanide poisoning andthe formation of methemoglobin e.g. by ingesting sodium nitrite orcertain other drugs and chemicals. The compounds described herein areused to prevent or treat symptoms of one or more of any of thesehypoxia-related conditions. In addition, the compounds attenuatehypoxia-induced cell necrosis and apoptosis, improve microvascularfunction during resuscitation from hemorrhagic shock, result inhemodynamic and oxygenation benefits during hypoxia (e.g. maintenance ofblood pressure and heart rate; preservation of microvascular blood flow;reduction in heart and brain hypoxia areas, etc.), and provideimprovement in several clinical symptoms, including reduced pain,decreased lactate dehydrogenase and/or RBC hemolysis, reduction indiastolic blood pressure, and an increase in blood oxygen levels(S_(p)O₂) during hypoxia challenge.

Methods of Making the Compounds

A generic scheme for making the compounds disclosed herein is depictedbelow in

where R1 and R2 are the same or different and are H; hydroxyl; halogen;or substituted or unsubstituted: alkyl, alkoxy, aryl, O-aryl,cycloalkane or heterocycle; R3 is an alkyl ester, aryl ester, analkylamide, an arylamide, phosphate or sulfate; M and Q are the same ordifferent and are O or (CH₂)n, where n=0-6 (e.g. 0, 1, 2, 3, 4, 5, or6); X, Y, Z, W and V are the same or different and are (independently)H, C, N, S or O; m=0-6 (e.g. 0, 1, 2, 3, 4, 5, or 6).

In this exemplary reaction, step (a) is conducted in the presence ofN-bromosuccinimide (NBS), α,α′-Azoisobutyronitrile (AIBN), and CCl₄, ata temperature in the range of from about 45-75° C., for about 5 h. (e.g.from about 1-10, 2-9, 3-8, 4-8, or 5 or 6 hours); and step (b) isconducted in the presence of K₂CO₃ and anhydrous DMF, room temperature,for about 5-10 h.

A generic scheme for making the promoiety-containing compounds disclosedherein is depicted below in Scheme II:

where R1 and R2 are the same or different and are H; hydroxyl; halogen;or substituted or unsubstituted: alkyl, alkoxy, aryl, O-aryl,cycloalkane or heterocycle; R3 is an alkyl ester, aryl ester, analkylamide, an arylamide, phosphate or sulfate; R4 is linear or branchedC1-C5 alkyl; M and Q are the same or different and are O or (CH₂)n,where n=0-6 (e.g. 0, 1, 2, 3, 4, 5, or 6); X, Y, Z, W and V are the sameor different and are (independently) H, C, N, S or O; m=0-6 (e.g. 0, 1,2, 3, 4, 5, or 6).

A typical synthesis will involve the condensation of the aldehydes, 2with equimolar of L-cysteine ethyl ester, 2 in the presence ofN-ethyldiisopropylamine hydrochloride (EDA) or triethyl amine (NET₃) toafford the ethyl ester prodrug, 3. Hydrolysis of the ethyl esterprotecting group of 3 with sodium hydroxide will yield the acid prodrug,4.

In some exemplary aspects, the compounds include the “PP” series ofcompounds and are made by the following synthetic Scheme III:

where X, Y and Z=C or N and R=—OCH₃ or —OH. In this exemplary reaction,step (a) is conducted in the presence of N-bromosuccinimide (NBS),α,α′-Azoisobutyronitrile (AIBN), and CCl₄, at a temperature in the rangeof from about 45-75° C., for about 5 h. (e.g. from about 1-10, 2-9, 3-8,4-8, or 5 or 6 hours); and step (b) is conducted in the presence ofK₂CO₃ and anhydrous DMF, room temperature, for about 5-10 h.

It is to be understood that this invention is not limited to particularembodiments described herein above and below, and as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

Where a range of values is provided, it is understood that eachintervening value between the upper and lower limit of that range (to atenth of the unit of the lower limit) is included in the range andencompassed within the invention, unless the context or descriptionclearly dictates otherwise. In addition, smaller ranges between any twovalues in the range are encompassed, unless the context or descriptionclearly indicates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Representative illustrativemethods and materials are herein described; methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the present invention.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference, and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual dates of publicavailability and may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as support for the recitation in the claims of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitations, such as “wherein [a particular feature or element] isabsent”, or “except for [a particular feature or element]”, or “wherein[a particular feature or element] is not present (included, etc.) . . .”.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

EXAMPLES Example 1

We tested whether derivatizing the alcohol moiety of vanillin into arylor alkyl amides and esters or inorganic phosphate esters would improvethe pharmacologic properties of these compounds. First the substitutionswould slow or make the compounds resistant to metabolism and lead tosignificantly sustained and enhanced pharmacologic activities. Inaddition, bulky ester or amide moieties would increase interactions withHb with a concomitant increase in Hb oxygen affinity and lead toincreased antisickling potency. The increased interactions would alsoslow down dissociation of the compound from the Hb and limit extensivemetabolism of the free aromatic aldehyde. The increased hydrophobicityof the compound would also lead to increased partitioning of thecompound into red blood cells and reduce off-target toxicity.Importantly, the bulky moiety on the pyridine ring would increase theinteraction with the F helix subunit and lead to stereospecificinhibition of polymer formation, resulting in the compounds exhibiting asecond antisickling mechanism of action that is independent of theprimary mechanism of increasing Hb affinity for oxygen. The F helix isvery important in stabilizing the polymer through secondary interactionbetween adjacent sickle Hb molecules, therefore any compound that bindsto the F helix and affect its conformation is expected to abrogate thisinteraction and weaken the polymer with concomitant antisicklingactivity. Consistently, the Hb variant Stanleyville (αAsn78↔αLsy78)inhibits polymerization.⁹

Experimental Section

Chemical Synthesis

General information: All reagents used in the synthesis and functionalassays were purchased from Sigma-Aldrich (St. Louis, Mo.) and ThermoFisher Scientific (Waltham, Mass.) and utilized without additionalpurification. GBT440 was purchased from MedChemExpress, LLC (MonmouthJunction, N.J.). ¹H-NMR and ¹³C-NMR spectra were obtained on a Bruker400 MHz spectrometer and tetramethylsilane (TMS) was used as an internalstandard. Peak positions are given in parts per million (δ). Columnchromatography was performed on silica gel (grade 60 mesh; BodmanIndustries, Aston, Pa.). Routine thin-layer chromatography (TLC) wasperformed on silica gel GHIF plates (250 μm, 2.5×10 cm; Analtech Inc.,Newark, Del.). MS spectra were obtained from a Perkin Elmer Flexar™UHPLC with AxION® 2 Time of Flight (TOF) Mass Spectrometer, and themolecular weight of the compounds was within 0.005% of calculatedvalues. Infrared spectra were obtained on Thermo Scientific™ Nicolet™iS10 FT-IR. Purity of the compounds was determined by HPLC using VarianMicrosorb™ 100-5 C18 column (250×4.6 mm), using Prostar 325 UV-Vis (210nm) as the detector. The HPC parameters used were: injection volume=15μL. sample concentration=3 mM, mobile phase=60MeCN-40H₂O, flow rate=1mL/min.

Statistical Analyses: All functional and biological assays evaluatingantisickling properties, Hb modification and oxygen affinity changeswere conducted in three biological replicates. Results are reported asmean values with standard deviations, from triplicate analyses.

Methyl 6-(bromomethyl)nicotinate

A mixture of methyl 6-methylnicotinate (1 eq) andα,α′-Azoisobutyronitrile (AIBN) (10%) was dissolved incarbontetrachloride (CCl₄). The solution was heated andN-bromosuccinimide (NBS) (1.1 eq) solution in CCl₄ was added drop wiseand refluxed for 5 hours. The reaction was cooled to room temperatureand the solvent evaporated. The mixture was then extracted usingdichloromethane and water followed by washing the organic layer withbrine. The organic layer was dried over sodium sulfate, filtered,evaporated and the crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=2:3 toobtain pure product as white powder and the yield was 66%. ¹H-NMR (400MHz, DMSO-d₆): δ 9.16 (d, J=1.56 Hz, 1H), 8.30 (dd, J=8.12, 2.2 Hz, 1H),7.53 (dd, J=8.12, 0.48 Hz, 1H), 4.58 (s, 2H), 3.96 (s, 3H). HRMS (ESI)m/z found 229.98 [M+H]⁺, Calculated 230.0586 [M]⁺.

Methyl 2-(bromomethyl)nicotinate

A mixture of methyl 2-methylnicotinate (1 eq) andα,α′-Azoisobutyronitrile (AIBN) (10%) was dissolved incarbontetrachloride (CCl₄). The solution was heated andN-bromosuccinimide (NBS) (1.1 eq) solution in CCl₄ was added drop wiseand refluxed for 5 hours. The reaction was cooled to room temperatureand the solvent evaporated. The mixture was then extracted usingdichloromethane and water followed by washing the organic layer withbrine. The organic layer was dried over sodium sulfate, filtered,evaporated and the crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=2:3 toobtain pure product as orange powder and the yield was 65%. ¹H-NMR (400MHz, DMSO-d₆): δ 8.71 (dd, J=4.8, 1.76 Hz, 1H), 8.28 (dd, J=7.92, 1.76Hz, 1H), 7.33 (m, 1H), 5.04 (s, 2H), 3.98 (s, 3H). HRMS (ESI) m/z found229.98 [M+H]⁺, Calculated 230.0586 [M]⁺.

Methyl 6-(bromomethyl)picolinate

A mixture of methyl 6-methylpicolinate (1 eq) andα,α′-Azoisobutyronitrile (AIBN) (10%) was dissolved incarbontetrachloride (CCl₄). The solution was heated andN-bromosuccinimide (NBS) (1.1 eq) solution in CCl₄ was added drop wiseand refluxed for 5 hours. The reaction was cooled to room temperatureand the solvent evaporated. The mixture was then extracted usingdichloromethane and water followed by washing the organic layer withbrine. The organic layer was dried over sodium sulfate, filtered,evaporated and the crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=2:3 toobtain pure product as white powder and the yield was 66%. ¹H-NMR (400MHz, DMSO-d₆): δ 8.05 (d, J=7.68 Hz, 1H), 7.99 (t, J=7.72 Hz, 1H), 7.77(d, J=7.68 Hz, 1H), 4.67 (s, 2H), 3.98 (s, 3H). HRMS (ESI) m/z found229.98 [M+H]⁺, Calculated 230.0586 [M]⁺.

Methyl 2-methylisonicotinate

A few drops of concentrated sulphuric acid were added to a solution ofmethylisonicotinic acid in methanol. The mixture was refluxed for 48hours. The resultant reaction mixture was neutralized with saturatedsodium bicarbonate solution followed by extraction with ethyl acetateand water. The organic layer was dried over sodium sulfate, filtered andsolvent evaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=4:1. Thepure compound was obtained as colorless oil with 96% yield. ¹H-NMR (400MHz, DMSO-d₆): δ 8.66 (d, J=5.08 Hz, 1H), 7.71 (s, 1H), 7.63 (dd,J=5.04, 0.32 Hz, 1H), 3.89 (s, 2H), 2.56 (s, 3H). HRMS (ESI) m/z found152.07 [M+H]⁺, Calculated 151.1626 [M]⁺.

Methyl 2-(bromomethyl)isonicotinate

A mixture of methyl 2-methylisonicotinate (1 eq) andα,α′-Azoisobutyronitrile (AIBN) (10%) was dissolved incarbontetrachloride (CCl₄). The solution was heated andN-bromosuccinimide (NBS) (1.1 eq) solution in CCl₄ was added drop wiseand refluxed for 5 hours. The reaction was cooled to room temperatureand the solvent evaporated. The mixture was then extracted usingdichloromethane and water followed by washing the organic layer withbrine. The organic layer was dried over sodium sulfate, filtered,evaporated and the crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=2:3 toobtain pure product as dark blue colored powder and the yield was 66%.¹H-NMR (400 MHz, DMSO-d₆): δ 8.77 (dd, J=5, 0.64 Hz, 1H), 8.02 (m, 1H),7.77 (dd, J=5, 1.56 Hz, 1H), 4.81 (s, 2H), 3.91 (s, 3H). HRMS (ESI) m/zfound 229.98 [M+H]⁺, Calculated 230.0586 [M]⁺.

Methyl 5-(bromomethyl)picolinate

A mixture of methyl 2-methylisonicotinate (1 eq) andα,α′-Azoisobutyronitrile (AIBN) (10%) was dissolved incarbontetrachloride (CCl₄). The solution was heated at 48° C. andN-bromosuccinimide (NBS) (1.1 eq) solution in CCl₄ was added drop wiseand stirred for 5 hours. The reaction was cooled to room temperature andthe solvent evaporated. The mixture was then extracted usingdichloromethane and water followed by washing the organic layer withbrine. The organic layer was dried over sodium sulfate, filtered,evaporated and the crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=2:3 toobtain pure product as white powder and the yield was 66%. ¹H-NMR (400MHz, DMSO-d₆): δ 8.78 (t, J=1.48 Hz, 1H), 8.06 (d, J=1.36 Hz, 2H), 4.82(s, 2H), 3.89 (s, 3H). HRMS (ESI) m/z found 229.98 [M+H]⁺, Calculated230.0586 [M]⁺.

Methyl 6-((2-formyl-4-methoxyphenoxy)methyl)nicotinate (PP1)

A mixture of 2-hydroxyl-5-methoxybenzaldehyde (1 eq) and methyl6-(bromomethyl)nicotinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 8-10 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=3:2 toobtain pure product as white powder with a yield of 82%. IR (Diamond,cm⁻¹): 2922, 2867, 1721, 1669, 1620, 1585, 1499, 1446, 1391, 1370, 1286,1224, 1174, 1141, 1121; ¹H-NMR (400 MHz, DMSO-d₆): δ 10.49 (s, 1H), 9.08(m, 1H), 8.36 (dd, J=8.16, 2.16 Hz, 1H), 7.80 (d, J=8.12 Hz, 1H), 7.25(m, 3H), 5.42 (s, 2H), 3.91 (s, 3H), 3.77(s, 3H); ¹³C-NMR (100 MHz,DMSO-d₆): δ 188.94, 165.00, 161.04, 154.62, 153.62, 149.59, 137.82,125.03, 124.77, 122.85, 121.31, 115.97, 110.78, 71.02, 55.59, 52.38.HRMS (ESI) m/z found 324.07 (M+Na)⁺, Calculated 301.2940 [M]⁺. Thepurity of the compound was checked by HPLC and was found to be 97% pure.

Methyl 2-((2-formyl-4-methoxyphenoxy)methyl)nicotinate (PP2)

A mixture of 2-hydroxyl-5-methoxybenzaldehyde (1 eq) and methyl2-(bromomethyl)nicotinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 8-10 hours. The solvent was then evaporated and thereaction mixture was extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=3:2 toobtain pure product as pale yellow powder with a yield of 82%. IR(Diamond, cm⁻¹): 2920, 1719, 1684, 1667, 1622, 1583, 1535, 1492, 1445,1404, 1372, 1276, 1215, 1168, 1142; ¹H-NMR (400 MHz, DMSO-d₆): δ 10.25(s, 1H), 8.74 (dd, J=4.76, 1.48 Hz, 1H), 8.23 (dd, J=7.8, 1.44 Hz, 1H),7.55 (m, 1H), 7.2 (m, 3H), 5.56 (s, 2H), 3.78 (s, 3H), 3.75(s, 3H);¹³C-NMR (100 MHz, DMSO-d₆): δ 188.73, 166.14, 155.39, 155.29, 153.46,151.55, 138.47, 126.17, 124.86, 123.66, 122.98, 116.13, 110.06, 70.98,55.61, 52.54. HRMS (ESI) m/z found 302.11 (M+H)⁺, 324.09 (M+Na)⁺,Calculated 301.2940 [M]⁺. The purity of the compound was checked by HPLCand was found to be 99% pure.

Methyl 6-((2-formyl-4-methoxyphenoxy)methyl)picolinate (PP3)

A mixture of 2-hydroxyl-5-methoxybenzaldehyde (1 eq) and methyl6-(bromomethyl)picolinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 8-10 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=3:2 toobtain pure product as white powder with a yield of 82%. IR (Diamond,cm⁻¹): 2953, 2851, 1720, 1682, 1618, 1585, 1494, 1396, 1369, 1296, 1222,1169, 1141; ¹H-NMR (400 MHz, DMSO-d₆): δ 10.47 (s, 1H), 8.03 (m, 2H),7.87 (d, J=7.32 Hz, 1H), 7.26 (m, 3H), 5.38 (s, 2H), 3.90 (s, 3H),3.76(s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆): δ 189.03, 164.99, 156.95,154.68, 153.59, 146.99, 138.57, 125.05, 125.01, 123.95, 123.84, 115.99,110.76, 71.08, 55.59, 52.39. HRMS (ESI) m/z found 302.10 (M+H)⁺, 324.08(M+Na)⁺, Calculated 301.2940 [M]⁺. The purity of the compound waschecked by HPLC and was found to be 98% pure.

Methyl 2-((2-formyl-4-methoxyphenoxy)methyl)isonicotinate (PP4)

A mixture of 2-hydroxyl-5-methoxybenzaldehyde (1 eq) and methyl2-(bromomethyl)isonicotinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 8-10 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=3:2 toobtain pure product as white powder with a yield of 82%. IR (Diamond,cm⁻¹): 2923, 1729, 1686, 1608, 1566, 1450, 1382, 1292, 1276, 1216, 1190,1171; ¹H-NMR (400 MHz, DMSO-d₆): δ 10.43 (s, 1H), 8.81 (dd, J=5, 0.56Hz, 1H), 8.02 (s, J=1.44 Hz, 1H), 7.80 (dd, J=5.04, 1.56 Hz, 1H), 7.24(m, 3H), 5.41 (s, 2H), 3.91 (s, 3H), 3.76 (s, 3H); ¹³C-NMR (100 MHz,DMSO-d₆): δ 188.81, 165.02, 157.80, 154.78, 153.63, 150.44, 137.79,125.09, 122.92, 121.82, 120.33, 116.25, 110.73, 71.27, 55.58, 52.81.HRMS (ESI) m/z found 302.10 (M+H)⁺, 324.08 (M+Na)⁺, Calculated 301.2940[M]⁺. The purity of the compound was checked by HPLC and was found to be98% pure.

Methyl 6-((2-formyl-3-hydroxyphenoxy)methyl)nicotinate (PP5)

A mixture of 2,6-dihydroxybenzaldehyde (1 eq) and methyl6-(bromomethyl)nicotinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 4 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=6:1 toobtain pure product as white powder with a yield of 58%. IR (Diamond,cm⁻¹): 3299, 2959, 1726, 1694, 1620, 1458, 1437, 1382, 1343, 1285, 1233,1172, 1122; ¹H-NMR (400 MHz, DMSO-d₆): δ 11.73 (s, 1H), 10.45 (s, 1H),9.09 (s, 1H), 8.34 (d, J=8.12, 1H), 7.81 (d, J=8.28 Hz, 1H), 7.51 (t,J=8.4 Hz, 1H), 6.67 (d, J=8.52 Hz, 1H), 6.56 (d, J=8.36 Hz) 5.41 (s,2H), 3.90 (s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆): δ 193.85, 165.39, 163.86,160.84, 160.41, 150.66, 138.40, 138.12, 125.47, 120.52, 111.05, 110.86,102.24, 70.91, 52.47. HRMS (ESI) m/z found 288.08 (M+H)⁺, 310.08(M+Na)⁺, Calculated 301.2940 [M]⁺. The purity of the compound waschecked by HPLC and was found to be 100% pure.

Methyl 2-((2-formyl-3-hydroxyphenoxy)methyl)nicotinate (PP6)

A mixture of 2,6-dihydroxybenzaldehyde (1 eq) and methyl2-(bromomethyl)nicotinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 4 hours. The solvent was then evaporated and thereaction mixture was extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=5:2 toobtain pure product as pale yellow powder with a yield of 58%. IR(Diamond, cm⁻¹): 2956, 1713, 1618, 1637, 1571, 1459, 1435, 1396, 1370,1287, 1238, 1170, 1141; ¹H-NMR (400 MHz, DMSO-d₆): δ 11.67 (s, 1H),10.19 (s, 1H), 8.75 (dd, J=4.8, 1.68 Hz, 1H), 8.25 (dd, J=7.84, 1.68 Hz,1H), 7.55 (m, 2H), 6.66 (d, J=8.2 Hz, 1H), 6.53 (d, J=8.4 Hz, 1H), 5.58(s, 2H), 3.79(s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆): δ 193.68, 166.15,162.26, 161.44, 155.02, 151.73, 138.75, 138.31, 126.02, 123.60, 110.66,109.49, 103.51, 70.59, 52.50. MS (ESI) m/z found 288.09 (M+H)⁺, 310.07(M+Na)⁺, Calculated 301.2940 [M]⁺. The purity of the compound waschecked by HPLC and was found to be 100% pure.

Methyl 2-((2-formyl-3-hydroxyphenoxy)methyl)isonicotinate (PP7)

A mixture of 2,6-dihydroxybenzaldehyde (1 eq) and methyl2-(bromomethyl)isonicotinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 4 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=5:2 toobtain pure product as pale yellow powder with a yield of 58%. IR(Diamond, cm⁻¹): 2961, 1733, 1716, 1639, 1620, 1577, 1460, 1441, 1373,1342, 1293, 1235, 1215, 1174; ¹H-NMR (400 MHz, DMSO-d₆): δ 11.68 (s,1H), 10.39 (s, 1H), 8.81 (dd, J=5, 0.6 Hz, 1H), 8.03 (d, J=0.56 Hz, 1H),7.81 (dd, J=5, 1.56 Hz 1H), 7.51 (t, J=8.4 Hz, 1H), 5.41 (s, 2H), 3.91(s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆): δ 193.51, 165.02, 162.37, 160.80,157.43, 150.43, 138.66, 137.84, 121.89, 120.38, 110.83, 109.74, 103.56,70.69, 52.83. MS (ESI) m/z found 288.09 (M+H)⁺, 310.07 (M+Na)⁺,Calculated 301.2940 [M]⁺. The purity of the compound was checked by HPLCand was found to be 99% pure.

Methyl 3-((2-formyl-4-methoxyphenoxy)methyl)picolinate (PP8)

A mixture of 2-hydroxyl-5-methoxybenzaldehyde (1 eq) and methyl3-(bromomethyl)picolinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 8-10 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=3:2 toobtain pure product as pale yellow powder with a yield of 82%. IR(Diamond, cm⁻¹): 2897, 1712, 1671, 1607, 1567, 1490, 1445, 1398, 1365,1275, 1232, 1165, 1139, 1106; ¹H-NMR (400 MHz, DMSO-d₆): δ 10.35 (s,1H), 8.65 (dd, J=4.6, 1.28 Hz, 1H), 8.2 (dd, J=7.84, 0.76 Hz, 1H), 7.66(dd, J=7.88, 4.68 Hz, 1H), 7.24 (m, 3H), 5.49 (s, 2H), 3.83 (s, 3H),3.77(s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆): δ 188.81, 166.12, 154.66,153.55, 148.49, 146.69, 136.89, 133.04, 126.43, 124.89, 122.96, 115.79,110.68, 67.44, 55.56, 52.34. MS (ESI) m/z found 324.08 (M+Na)⁺,Calculated 301.2940 [M]⁺. The purity of the compound was checked by HPLCand was found to be 98% pure.

Methyl 5-((2-formyl-4-methoxyphenoxy)methylpicolinate (PP9)

A mixture of 2-hydroxyl-5-methoxybenzaldehyde (1 eq) and methyl5-(bromomethyl)picolinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 8-10 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=3:2 toobtain pure product as white powder with a yield of 82%. IR (Diamond,cm⁻¹): 2962, 2864, 2767, 1719, 1684, 1673, 1588, 1489, 1458, 1395, 1355,1274, 1218, 1198, 1150, 1127; ¹H-NMR (400 MHz, DMSO-d₆): δ 10.40 (s,1H), 8.84 (d, J=1.36 Hz,1H), 8.10 (m, 2H), 7.28 (m, 2H), 7.2 (d, J=2.92Hz, 1H), 5.39 (s, 2H), 3.89 (s, 3H), 3.76 (s, 3H); ¹³C-NMR (100 MHz,DMSO-d₆): δ 188.77, 154.89, 154.57, 148.62, 135.93, 135.67, 135.93,135.67, 125.92, 125.17, 123.28, 114.92, 111.30, 68.52, 55.90, 52.97. MS(ESI) m/z found 302.10 (M+H)⁺, 324.09 (M+Na)⁺, Calculated 301.2940 [M]⁺.The purity of the compound was checked by HPLC and was found to be 98%pure.

Methyl 6-((2-formyl-3-hydroxyphenoxy)methyl)picolinate (PP10)

A mixture of 2,6-dihydroxybenzaldehyde (1 eq) and methyl6-(bromomethyl)picolinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 4 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=6:1 toobtain pure product as white powder with a yield of 58%. IR (Diamond,cm⁻¹): 3065, 3010, 2956, 2890, 1738, 1697, 1640, 1614, 1583, 1462, 1454,1400, 1358, 1297, 1242, 1190, 1171, 1150; ¹H-NMR (400 MHz, DMSO-d₆): δ11.74 (s, 1H), 10.44 (s, 1H), 8.04 (m, 2H), 7.91 (dd, J=7.48, 1.12 Hz,1H), 7.52 (t, J=8.4 Hz, 1H), 6.7 (d, J=8 Hz, 1H), 6.56 (d, J=8.4 Hz,1H), 5.39 (s, 2H), 3.90 (s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆): δ 193.79,164.99, 162.43, 160.69, 160.62, 149.59, 138.65, 137.84, 124.81, 121.26,110.79, 109.81, 103.42, 70.49, 52.41. MS (ESI) m/z found 288.09 (M+H)⁺,310.07 (M+Na)⁺, Calculated 301.2940 [M]⁺. The purity of the compound waschecked by HPLC and was found to be 100% pure.

Methyl 3-((2-formyl-3-hydroxyphenoxy)methyl)picolinate (PP11)

A mixture of 2,6-dihydroxybenzaldehyde (1 eq) and methyl3-(bromomethyl)picolinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 4 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=6:1 toobtain pure product as pale yellow powder with a yield of 54%. IR(Diamond, cm⁻¹): 3092, 2923, 2851, 1774, 1712, 1632, 1599, 1566, 1515,1478, 1366, 1276, 1231, 1180, 1136, 1072; ¹H-NMR (400 MHz, DMSO-d₆): δ11.72 (s, 1H), 10.32 (s, 1H), 8.65 (dd, J=4.64, 1.52 Hz, 1H), 8.22 (m,1H), 7.66 (dd, J=7.88, 4.64 Hz, 1H), 7.54 (t, J=8.4 Hz, 1H), 6.66 (d,J=8 Hz, 1H), 6.57 (d, J=8.4 Hz, 1H), 5.51 (s, 2H), 3.84 (s, 3H), ¹³C-NMR(100 MHz, DMSO-d₆): δ 193.59, 166.09, 162.46, 160.68, 148.53, 146.54,138.72, 136.83, 132.78, 126.44, 110.74, 109.79, 103.26, 67.04, 52.35. MS(ESI) m/z found 310.08 (M+Na)⁺, Calculated 301.2940 [M]⁺. The purity ofthe compound was checked by HPLC and was found to be 100% pure.

Methyl 5-((2-formyl-4-methoxyphenoxy)methyl)nicotinate (PP12)

A mixture of 2-hydroxyl-5-methoxybenzaldehyde (1 eq) and methyl5-(bromomethyl)nicotinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was refluxed at roomtemperature for 8-10 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=3:2 toobtain pure product as white powder with a yield of 82%. IR (Diamond,cm⁻¹): 2969, 1718, 1670, 1602, 1572, 1498, 1435, 1405, 1382, 1279, 1217,1185, 1160, 1119; ¹H-NMR (400 MHz, DMSO-d₆): δ 10.37 (s, 1H), 9.06 (d,J=2 Hz, 1H), 8.97 (d, J=2.04 Hz, 1H), 8.42 (t, J=2.04 Hz, 1H), 7.29 (m,2H), 7.2 (d, J=3.08 Hz, 1H), 5.38 (s, 2H), 3.9 (s, 3H), 3.77(s, 3H);¹³C-NMR (100 MHz, DMSO-d₆): δ 188.87, 165.04, 154.64, 153.64, 152.90,149.49, 135.99, 132.63, 125.44, 125.13, 122.88, 116.21, 110.71, 67.83,55.58, 52.49. MS (ESI) m/z found 324.08 (M+Na)⁺, Calculated 301.2940[M]⁺. The purity of the compound was checked by HPLC and was found to be99% pure.

Methyl 5-((2-formyl-3-hydroxyphenoxy)methyl)nicotinate (PP13)

A mixture of 2,6-dihydroxybenzaldehyde (1 eq) and methyl5-(bromomethyl)nicotinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 4 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=6:1 toobtain pure product as white powder with a yield of 54%. IR (Diamond,cm⁻¹): 2907, 1719, 1643, 1617, 1584, 1460, 1429, 1397, 1370, 1292, 1246,1179, 1152, 1121; ¹H-NMR (400 MHz, DMSO-d₆): δ 11.72 (s, 1H), 10.33 (s,1H), 9.05 (d, J=1.8 Hz, 1H), 8.99 (d, J=1.76 Hz, 1H), 8.43 (t, J=2 Hz,1H), 7.55 (t, J=8.2 Hz, 1H), 6.73 (d, J=8.2 Hz, 1H), 6.56 (d, J=8.44 Hz,1H), 5.39 (s, 2H), 3.91(s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆): δ 193.74,176.82, 165.04, 162.40, 160.72, 152.87, 149.53, 138.63, 135.99, 132.32,110.80, 109.76, 103.42, 67.28, 52.49. MS (ESI) m/z found 288.08 (M+H)⁺,Calculated 301.2940 [M]+. The purity of the compound was checked by HPLCand was found to be 99% pure.

Methyl 5-((2-formyl-3-hydroxyphenoxy)methyl)picolinate (PP14)

A mixture of 2,6-dihydroxybenzaldehyde (1 eq) and methyl5-(bromomethyl)picolinate (1 eq) was dissolved in anhydrousN,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K₂CO₃) (1.2eq) was added to this mixture and the reaction was stirred at roomtemperature for 4 hours. The solvent was then evaporated and thereaction mixture extracted with ethyl acetate and water. The organiclayer was dried over sodium sulfate, filtered and the solventevaporated. The crude product was purified using SiO₂ columnchromatography and eluted with the solvent system EtOAc:hexanes=6:1 toobtain pure product as white powder with a yield of 54%. IR (Diamond,cm⁻¹): 2954, 1731, 1679, 1644, 1618, 1574, 1458, 1440, 1392, 1357, 1289,1249, 1177, 1144, 1122; ¹H-NMR (400 MHz, DMSO-d₆): δ 11.74 (s, 1H),10.37 (s, 1H), 8.86 (s, 1H), 8.12 (m, 2H), 7.54 (t, J=8.4 Hz, 1H), 6.72(d, J=8.32 Hz, 1H), 6.56 (d, J=8.4 Hz, 1H), 5.40 (s, 2H), 3.89 (s, 3H);¹³C-NMR (100 MHz, DMSO-d₆): δ 193.83, 162.42, 160.69, 148.72, 147.01,138.64, 136.32, 135.91, 135.78, 124.64, 109.79, 103.37, 67.31, 52.39. MS(ESI) m/z found 310.08 (M+Na)⁺, Calculated 301.2940 [M]⁺. The purity ofthe compound was checked by HPLC and was found to be 99% pure.

Blood Collection and Purification of Hemoglobin

Human blood samples were obtained from healthy volunteers at VirginiaCommonwealth University. The use of the biological samples was reviewedand approved by the IRB, in accordance of institutional regulations.Leftover blood samples from patients with homozygous SS were obtainedand utilized, based on an approved IRB protocol at the Children'sHospital of Philadelphia, with informed consent. Hemoglobin was purifiedfrom normal human blood samples according to the literature procedure.¹⁰

Oxygen Equilibrium Curve Studies

Oxygen equilibrium curve (OEC) studies to determine the effect of thecompounds on Hb affinity for oxygen were conducted following standardprocedure.^(8,11) Whole blood (30% hematocrit) was incubated in theabsence or presence of compounds (0.5 mM, 1.0 mM and 2.0 mM) solubilizedin DMSO. The blood-compound reaction samples were then incubated in anIL 237 tonometer (Instrumentation Laboratories, Inc., Lexington, Mass.)for about 7 min at 37° C. under a gas mixture containing O₂ atconcentrations of 0.804%, 2.935% and 5.528% and allowed to equilibrateat O₂ tensions of 6, 20 and 40 mmHg. After equilibration, the sample wasremoved via syringe and aspirated into an ABL 700 series table topautomated blood gas analyzer (Radiometer America, Inc., Westlake, Ohio)to determine total hemoglobin (tHb), hematocrit (Hct), pH, pCO₂, partialpressure of oxygen (pO₂), and the Hb-O₂ saturation (sO₂) values. Themeasured values of pO₂ and sO₂ at each oxygen saturation level were thensubjected to nonlinear regression analysis using the software Scientist(Micromath, Salt Lake City, Utah) with the following equation:

${{sO}_{2}\%} = {100 \times \frac{{pO}_{2}^{N}{mmHg}}{{P_{50}^{N}({mmHg})} + {{pO}_{2}^{N}({mmHg})}}}$

This equation was used to calculate P₅₀ and Hill coefficient (N) values.ΔP₅₀ (%) was determined as:

${\Delta{P_{50}(\%)}} = {100 \times \frac{{P_{50}{of}{lysates}{from}{untreated}{cells}} - {P_{50}{of}{lysates}{from}{treated}{cells}}}{P_{50}{of}{lysates}{from}{unrteated}{cells}}}$

Adduct Formation Studies Using Normal Whole Blood and Hemoglobin

Time dependent and concentration dependent Hb-compound adduct formationstudies were performed with whole blood using standard procedures. In a96-well deep well plate (Thermo Scientific), 0.5 mM, 1.0 mM and 2.0 mMconcentrations of the compounds were added to 600 μL of whole blood (30%hematocrit) and/or Hb (1.56 mM). These were incubated at 37° C. for 24 hwith shaking (at 140 rpm). At 1, 4, 8, 12 and 24 h time intervals, 50 μLaliquots of this mixture were removed from each well using amultichannel pipette and added to respective tubes containing 50 μL ofsodium cyanoborohydride (NaBH₃CN) and sodium borohydride (NaBH₄) mixture(1:1 v/v 50 mM stock) to terminate the Schiff-base reaction, fix theSchiff-base adducts and reduce the free reactive aldehyde.¹² Aftermixing, the tubes were stored immediately at −80° C. until ready forbatch analysis by cation-exchange HPLC (Hitachi D-700 Series, HitachiInstruments, Inc. San Jose, Calif.).^(5,6) The observed Hb adducts,calculated as percentages (%) of Hb modification were plotted as afunction of time (h); peak areas were quantitated using the HPLCsoftware.

Hemoglobin Modification, Oxygen Equilibrium and Antisickling StudiesUsing Human Sickle Blood

Blood suspensions from subjects with homozygous SCD (resuspended inHemox™ buffer to a final hematocrit 20%) were incubated under air in theabsence or presence of the compounds (0.5 mM, 1 mM and 2 mM) at 37° C.for 1 h to ensure that binding had attained equilibrium. The suspensionswere then incubated under hypoxic conditions (2.5% oxygen/97.5%nitrogen) at 37° C. for 1 h. Aliquot samples were fixed with 2%glutaraldehyde solution without exposure to air, and then subjected tomicroscopic morphological analysis of bright field images (at 40×magnification) of single layer cells on an Olympus BX40 microscopefitted with an Infinity Lite B camera (Olympus), and the coupled ImageCapture software. The residual samples were washed in phosphate-buffersaline, and hemolyzed in hypotonic lysis buffer for subsequentHb-modification and oxygen equilibrium analyses. 8,12

For oxygen equilibrium studies, approximately 100 μL aliquot samplesfrom the above clarified lysate were added to 4 mL of 0.1M potassiumphosphate buffer, pH 7.0, in cuvettes and subjected to hemoximetryanalysis using a Hemox™ Analyzer (TCS Scientific Corp.) to assess P₅₀shifts. Degree of P₅₀ shift (ΔP₅₀) was expressed as percentage fractionsof control DMSO-treated samples.

${{\Delta P}_{50}(\%)} = {100 \times \frac{\begin{matrix}{{P_{50}{of}{lysates}{from}{untreated}{cells}} -} \\{P_{50}{of}{lysates}{from}{treated}{cells}}\end{matrix}}{P_{50}{of}{lysates}{from}{untreated}{cells}}}$

Finally, for the Hb adduct formation studies, the above clarifiedlysates were subjected to cation-exchange HPLC (Hitachi D-7000 Series,Hitachi Instruments, Inc., San Jose, Calif.), using a weakcation-exchange column (Poly CAT A: 30 mm×4.6 mm, Poly LC, Inc.,Columbia, Md.). A commercial standard consisting of approximately equalamounts of composite HbF, HbA, HbS and HbC (Helena Laboratories,Beaumont, Tex.), was utilized as the reference for isotypes. The areasof new peaks, representing HbS adducts, were obtained, calculated aspercentage fractions of total Hb area, and reported as levels ofmodified Hb.

Antisickling Activities of Compounds Under 100% Nitrogen

To demonstrate potential non Hb-O₂ affinity-dependent activities of themolecules, we tested the antisickling properties of selected compounds(PP9 and PP14) under complete deoxygenated conditions. We used TD7 andGBT440 as reference controls; the latter is also an aromatic aldehydewith the most potent reported in vitro p50 shifts and antisicklingproperties, and is currently in phase III clinical studies for thetreatment of SCD. Briefly, antisickling studies were conducted aspreviously described, using 2 mM concentrations of the test molecules.After 1 h, aliquot samples were fixed with 2% glutaraldehyde withoutexposure to air. Then the incubation chamber was opened and exposed toair for 15 minutes to ensure complete re-oxygenation and reversal of thesickled cells to normal round cells. Reversal was confirmed bymicroscopy. The incubation chamber was then closed and the assay wasrepeated under 100% nitrogen gas for 30 minutes, at which point aliquotswere obtained and fixed. Aliquot samples were then subjected tomicroscopic morphological analysis of bright field images (at 40×magnification) of single layer cells on an Olympus BX40 microscopefitted with an Infinity Lite B camera (Olympus), and the coupled ImageCapture software. Resulting sickled cells (percentages) were comparedacross samples, and between aliquots of the same samples that had beenobtained either under 2.5% oxygen or 100% nitrogen. This experimentaldesign utilized aliquots of the same samples under different gasconditions, and thereby ensured that the assay was free of any potentialerrors associated with variability in hematocrit and accuracy in addingthe molecules.

X-ray Crystallography

Freshly made solutions of compounds in DMSO were added to deoxygenated(deoxy) Hb (30 mg/mL protein) at an Hb tetramer-compound ratio of 1:10.Then, the complex mixture was saturated with carbon monoxide and allowedto incubate for 2 h to form COHb-compound complexes. Sodiumcyanoborohydride (NaBH₃CN) was then added to this mixture to reduce theSchiff-base adduct formed between the protein and compound to thecorresponding irreversible alkylamine covalent bond. The resultingsolution was crystalized using 10-20% PEG 6000, 100 mM HEPES buffer, pH7.4 using the batch method as previously published.¹² Single cherry redneedle crystals were formed in 1-3 days and were used to collect x-raydiffraction data at 100 K using Rigaku MicroMax™ 007HF X-ray Generator,Eiger R 4M Detector and Oxford Cobra Cryo-system (The Woodlands, Tex.).The crystals were first cryoprotected with 80 μL mother liquor mixedwith 62 μL of 50% PEG6000. The dataset was processed with the d*treksoftware (Rigaku) and the CCP4 suite of programs.¹³ The crystalstructures of the COHb-compound complexes were determined by a molecularreplacement method with Phenix program,¹⁴ using the native R2-statecrystal structure (PDB ID 1BBB) as a search model. The structures wererefined using both Phenix and CNS while model building and correctionwas carried out using COOT.^(15,16)

In Vivo Pharmacologic Effect of PP Compounds in Mice

We tested in vivo pharmacologic (pharmacodynamics) effects of select PPcompounds in C57BL/6 mice. The animals were treated with a singleintraperitoneal (IP) (150 mg/kg body weight) dose of PP6, PP10, PP14,and TD7, which were formulated with 30% PEG300. Blood samples wereobtained prior to treatment (0) and 1, 3 and 6 h post-treatment, viasubmandibular bleeding, and subjected to hemolysis using standardmethods. Clarified lysates, free of cell debris and red blood cellghosts were analyzed by cation-exchange HPLC to determine the levels ofdrug-modified hemoglobin (adducts); and to conduct oxygen equilibriumstudies to determine degrees of shift in p50 values using methodologiesdescribed earlier for in vitro studies on human samples.^(8,12)

In a subsequent follow-up experiment, a new excipient obtained fromCatalent Pharma Solutions was also used to formulate PP14 and used for asingle intraperitoneal (IP) and oral (150 mg/kg body weight) dose. Bloodsamples were obtained prior to treatment (0) and 1, 3 or 5 hpost-treatment, via submandibular bleeding, and used to determine thelevels of drug-modified hemoglobin (adducts); and to conduct oxygenequilibrium studies to determine degrees of shift in p50 values asdescribed above.

Results and Discussion Chemical Synthesis

A representative general scheme for synthesizing “PP” compounds is shownin Scheme III (above) and representative compounds PP1, PP2, PP3, PP4,PP5, PP6, PP7, PP8, PP9, PP10, PP11, PP12, PP13, and PP14 are depictedin FIG. 2. All compounds were used for functional and biological invitro studies. Selected compounds were used for in vivo and structuralstudies.

Structural Study Showed PP Compounds Bind to the α-Cleft of Hemoglobin

Based on crystallographic binding of vanillin and TD7 to Hb, westructurally modified the TD compounds into PP compounds to increaseinteractions with Hb, as well as make closer contacts with the F helix.Several PP compounds were crystallized with Hb and with CO-liganded Hband the crystal structures of several of these complexes in the R2-stateconformation were successfully determined. The structure of PP9 whichhas been refined to 1.9 Å is described here, noting that the othercomplexes show similar binding, although differences also occur thatmight explain differences in their functional activities.

The overall tetrameric structure of PP9 is indistinguishable (rmsd ˜0.4Å) from the native R2 structure (1BBB) or the R2 structure in complexwith 5-HMF (PDB code 1QXE) or in complex with TD7. As observed withthese compounds, a pair of PP9 covalently bound to the N-terminal αVal1amines (Schiff-base interaction) in a symmetry-related fashion is seenat the α-cleft as shown in FIG. 3 for PP9. Formation of the Schiff baseinteraction between the aromatic aldehyde and the αVal1 nitrogen at theα-cleft of Hb directed the ortho-substitutedpyridinylmethoxy-methylester upwards towards the surface of the α-cleftand close to the αF helix. Since both molecules bound in a symmetricalfashion, detailed interactions of Hb focus on α2Val1 binding PP9. Thebenzaldehyde ring makes both intra- and inter-subunit hydrophobicinteractions with α2Ala130, α2Ser131, α2Thr134 and α1Thr134. The twopyridine rings from the two PP9 molecules make face-to-face π-π stackinginteractions (3.5 Å and greater) with each other. Interestingly, thepyridinylmethoxy-methylester group is oriented toward the αF helix whencompared to the hydroxyl moiety of TD7. In fact, thepyridinylmethoxy-methylester makes intra-subunit hydrophobicinteractions with α2Pro77 of the αF helix (3.1 Å and greater) comparedto the 3.5 Å observed with TD7. In addition, the meta-located pyridinenitrogen and the oxygen of the ester also make extensive and very strongintra-subunit water-mediated interactions with the backbone atoms ofα2Val73, α2Asp75 and α2Met76 of the αF helix. These interactions aremissing in the TD7 crystal structure. In summary, PP9 binds in asymmetry-related fashion; making several intra- and inter-subunitinteractions that leads to stabilization of the R-state Hb, increasesthe protein affinity for oxygen, and concomitantly reduceshypoxia-induced polymerization. Importantly, the close interactions withthe αF helix translate into significant perturbation of the helix andresult in polymer destabilization. Finally, the many and intricateinteractions with the protein (missing in TD7) stabilize the Schiff-baseadduct and reduce dissociation of the bound compound.

In summary, replacing the hydroxyl group with an ester leads to novelcompounds that (1) bind strongly to Hb and exhibit increased functionaland biological effects; (2) increase strong interactions with the Fhelix that translate into strong perturbation of the helix and lead topolymer destabilization, leading to a second mechanism of antisickling;and (3) reduce dissociation of the bound compound, thus reducingmetabolism. These observations are consistent with functional andbiological data.

PP compounds increased Hb affinity for oxygen in normal whole blood invitro Aromatic aldehydes are known to prevent hypoxia-induced Hb Spolymerization by increasing the Hb affinity for oxygen.^(1,2,11,12) Wetherefore tested the PP compounds in a dose-dependent manner (0.5, 1, 2mM) for their effect on Hb oxygen binding property. Vanillin and TD7were tested as controls. The results are summarized in FIG. 4. The studyshowed a dose dependent effect for all compounds, with PP2, PP6, PP7,PP9, PP12, and PP14 showing the most significant activities. PP1 and PP4showed minimal effect because of solubility issue. TD7 showed relativelysimilar potency as most of the best PP compounds although lower than ourmost potent PP compounds, while vanillin as expected showed very weakfunctional effect. These findings confirmed that the potent in vitroactivity previously seen in TD7 was at least conserved in the novel PPcompounds.

PP Compounds Exhibited Extended Functional Effect (Hb Modification) inVitro

Aromatic aldehydes that increase Hb affinity for oxygen with aconcomitant antisickling effect do so by using the aldehyde moiety tobind to the N-terminal αVal1 nitrogens of Hb, forming Schiff-baseadducts.^(1,2,11,12) Schiff-base adducts can be accurately quantified byHPLC (Hb modification or Hb adduct). To test the degree and duration ofHb modification, we incubated 2 mM concentrations of PP compounds, andthe controls vanillin and TD7 with normal adult blood, hematocritadjusted to 30% at 37° C. for 24 h. At defined time points (1, 4, 8, 12and 24 h) aliquot samples were drawn, mixed with a solution containingsodium borohydride/cyanoborohydride to fix the Schiff-base, andsubsequently analyzed by cation-exchange HPLC. The results in FIG. 5showed that Hb modification (adduct formation) was sustained for theentire 24 h experimental period with most of the PP compounds. Thisobservation suggests decreased metabolism of the compounds in wholeblood that is partly due to the added structural modifications to thecompounds that prevented or reduced enzymatic metabolism, and partly dueto strong interactions with the protein to form a more stabilizedSchiff-base adduct, thus reducing dissociation of the compound. Incontrast, TD7, after reaching a maximum effect at 1 hr, graduallydecreased in potency and at 24 hours had lost 45% of its activity,consistent with the low bioavailability of this compound. Also and asexpected, vanillin showed no effect after 4hrs.

As noted above, a Schiff-base interaction is an equilibrium betweenbound and unbound complex, and when unbound the compound can potentiallybe metabolized into a pharmacologically inactive non-aldehyde.Therefore, for formation of a stable Schiff-base complex and thus aneffective pharmacologic outcome, aromatic aldehydes would make stronginteractions to Hb, and exhibit a slow rate of dissociation. Ourstructural studies clearly show that binding between PP9 and Hb isstronger than binding between TD7 and Hb, suggesting that PP9dissociates slowly from the bound protein when compared to TD7, andconsistent with the results of the time-dependent Hb modificationstudies. Similar strong interactions with the protein, and particularlywith the F helix, are also observed for several of the PP compounds.These findings validate the novelty of these compounds as they clearlysuggest an improvement in the metabolic profile of the PP compounds whencompared to TD7. As discussed below, these in vitro findings translatedinto improved PD/PK properties in vivo.

PP Compounds Modified Sickle Hb, Increased Its Affinity for Oxygen andPrevented Hypoxia-Induced Erythrocyte Sickling in Vitro.

The studies described above were conducted using normal whole blood.Next, we investigated whether the improvement in Hb oxygen affinity andthe compounds sustained activity also translated to similar effects inSS blood from SCD patients. The PP compounds with the control vanillinand TD7 were therefore tested using sickle RBCs for their effect on Hbmodification, Hb oxygen affinity and sickling at 0.5, 1 and 2 mM. Theresults are summarized in FIGS. 6-8. As observed with normal wholeblood, the compounds potently increased Hb affinity for oxygen, Hbmodification and inhibition of RBC sickling in a dose-dependent fashion.At the lowest concentration of 0.5 mM, four of the compounds, PP2, PP6,PP8, and PP14 showed more than 35% sickling inhibition cinoared to 26%by TD7. At 1 mM, PP8 and PP9 inhibited nearly 100% of sickling, whilePP2, PP6, PP14 inhibited sickling by more than 70%. This compares with47% inhibition by TD7 at 1 mM. Expectedly, vanillin showed only minimalsickling inhibition. It is clear that the modifications of the presentcompounds has led to significant improvement in antisickling activity.For a disease that requires relatively high concentrations of drug toreach a therapeutic window, a 2-fold increase in potency is verysignificant, and this fact, taken in conjunction with the fact that thePP compounds show superior PK properties, makes these compounds noveland highly worth developing.

Also noteworthy and significant is the observation that while TD7 andsome of the PP compounds demonstrated an almost linear correlationbetween their ability to increase Hb oxygen affinity and antisicklingactivity, others, such as PP2, PP3, PP6, PP8, PP9 and PP14 showed a weakcorrelation between these parameters. In fact, these compoundsdemonstrated the most potent antisickling effect despite only marginallyincreasing Hb affinity for oxygen. This observation is consistent withthese compounds exhibiting multiple mechanisms of antisickling activitythat include increasing the oxygen affinity of Hb, and directstereospecific polymer destabilization; the latter is validated by thecrystal structures that show that PP9 and several of the PP compoundsmake significant hydrophobic and hydrogen-bond interactions with the Fhelix. As discussed below, F helix perturbation leads to these compoundsinhibiting RBC sickling even at 100% nitrogen, which is independent ofthe primary antisickling mechanism of increasing Hb oxygen affinity.

PP Compounds Inhibit RBC Sickling Under 100% Nitrogen

Placing a bulkier group e.g. ester or amide moiety on the pyridinemoiety results in compounds that behave differently than previousaromatic antisickling aldehydes. The resulting molecules make closerinteractions with the surface located αF helix and lead tostereospecific inhibition of polymer formation. Thus, the compoundsexhibit a second antisickling mechanism of action (an Hb-O₂ affinityindependent antisickling mechanism), and this can be followed byconducting the antisickling assay at 100% nitrogen. Selected PPcompounds were therefore tested using sickle RBCs for their antisicklingeffect at 100% nitrogen. TD7 and GBT440 were used as positive controls.GBT440 is also an antisickling aromatic aldehyde that is currently inphase III clinical studies for the treatment of SCD (clinicaltrials.gov,NCT03036813). GBT440 is highly potent in preventing hypoxia-inducedpolymerization and concomitant RBC sickling, and has shownproof-of-concept in animal models and humans. As indicated earlier, forinternal control purposes, the same samples were used for testing theantisickling effect of these compounds with 2.5% oxygen prior to testingunder nitrogen gas, therefore helping elucidate the antisickling effectsdue to increased Hb affinity for oxygen. The results are summarized inFIG. 9, and clearly showed that at a 2 mM concentration, the compoundsnot only inhibit sickling via the primary mechanism of action ofincreasing Hb oxygen affinity, but they also showed a novel mechanism ofaction by inhibiting sickling under 100% nitrogen, unlike othermolecules. The biological data is consistent with the structural data,indicating that these compounds are able to interact very strongly(hydrophobic and hydrogen-bonding) with the surface located αF helix ofHb, leading to destabilization of the polymer with a concomitantantisickling activity that is independent of oxygen affinity for Hb.

PP Compounds Showed Improved Pharmacodynamic Activities in Vivo

Three of the PP compounds (PP6, PP10 and PP14) with high and sustainedfunctional/biological functional/biological activities were selected foran assessment of their in vivo pharmacologic (pharmacodynamic) effectsusing wild-type C57BL/6 mice. Excipients for formulating the compoundswere optimized.

In an earlier experiment, using 30% PEG300 to formulate the testcompounds, the animals were treated with a single intraperitoneal (IP)150 mg/kg body weight) dose of PP6, PP10, PP14. All three selected PPcompounds demonstrated significant in vivo modification of intracellularHb in mice after IP administration, with increasing levels from 1 h tothe 6 h experimental period (n=2 mice per compound, FIG. 10A). Micetreated with PP14 demonstrated the highest levels of modified Hb(19.1±0.6% at 6 h), compared to 16.9±1.4% and 9.6±1.9% for PP6-treatedand PP10-treated mice, respectively. Conversely, only 1.5±0.3% modifiedHb was observed in mice treated with TD7. All three PP compounds led toa significant increase in Hb affinity for oxygen when compared to TD7.Corresponding changes in oxygen affinity (Δp50) were observed at themeasured time points of 3 h and 6 h, compared to 0 h pre-treatmentsamples (FIG. 10B).

Using the 30% PEG300 excipient did not give a uniform dispersion andthis could affect bioavailability and the PD effect. We thereforeexperimented with several different excipients for formulating one ofthe compounds, PP14, for testing. The optimal excipient, which gaveuniform compound dispersions, was obtained from Catalent PharmaSolutions. Consistent with the optimized formulation, we observed asignificant increase in PD effect. At 150 mg/kg, mice treated with PP14via IP injection showed significantly higher Hb modifications of21.8±1.6% (1 hr), 37.7±5.0% (3 hr) and 51.1±4.3% (5 hr), respectively(FIG. 11A). This compares to Hb modifications of 7% (1 hr), 13% (3 hr)and 19% (6 hr), respectively by PP14 when 30% PEG300 was used as theexcipient (FIG. 10A). The high Hb modification also resulted insignificant increases in Hb affinity of 43.5±7.7% at 5 hr (FIG. 11B).This compares to 12% using the excipient 30% PEG300 (FIG. 10B). Clearlyoptimizing the formulation led to a 3-4 fold more potent compoundactivity. Using oral gavage with the same excipient also resulted inhigh levels of modified Hb at 14.5±3.7% (1 hr), 16.3±2.2% (3 hr) and17.1±4.4% (5 hr), as well as corresponding increase in Hb oxygenaffinity of 10.3±1.5% (FIG. 11).

The study demonstrates the superiority of the PP compounds compared toTD7 and vanillin, which is likely attributable to the structuralmodifications described herein.

Conclusion

We have developed new compounds to overcome the disadvantages ofpredecessor molecules. The compounds exhibit several advantageousattributes: first, the structural modifications allow the compounds tomake very strong interactions with the surface located αF helix of Hb,which is known to be important in stabilizing the polymer. Theinteractions with the F helix destabilize the polymer, resulting in thecompounds exhibiting an independent and novel Hb-O₂ antisicklingmechanism of action, in addition to the primary Hb-O₂ dependentantisickling mechanism. This dual mechanism translated into very potentand significantly enhanced antisickling activities. Moreover, the Hb-O₂independent antisickling mechanism permits the prevention of sicklingwithout drastically changing oxygen delivery capabilities, making thecompounds more useful in treating SCD compared to other related aromaticaldehydes. This is critical for a disease that is characterized bysevere hypoxia. Second, due to the structural modifications, thecompounds are resistant to metabolism due to enhanced binding tohemoglobin, which stabilizes the Schiff-base adduct and leads tosignificantly sustained and improved pharmacologic activities in vitroand in vivo. These attributes are critical properties for a regularlyadministered agent to treat SCD—as it is chronic condition.

References for Example 1

-   1. Safo, M. K.; Ahmed, M. H.; Ghatge, M. S.; Boyiri, T.    Hemoglobin-ligand binding: Understanding hb function and allostery    on atomic level. Biochim. Biophys. Acta. 2011, 1814, 797-809.-   2. Pauling, L.; Itano, H. A. Sickle cell anemia, a molecular    disease. Science 1949, 109, 443.-   3. Cretegny, I.; Edelstein, S. J. Double strand packing in    hemoglobin S fibers, J. Mol. Biol. 1993, 230, 733-738.-   4. Habara, A.; Steinberg, M. H. Minireview: Genetic basis of    heterogeneity and severity in sickle cell disease. Exp. Biol. Med.    (Maywood) 2016, 241, 689-696.-   5. Charache, S.; Terrin, M. L.; Moore, R. D.; Dover, G. J.;    McMahon, R. P.; Barton, F. B.; Waclawiw, M.; Eckert, S. V. Design of    the multicenter study of hydroxyurea in sickle cell anemia.    investigators of the multicenter study of hydroxyurea. Control.    Clin. Trials. 1995, 16, 432-446.-   6. Abraham, D. J.; Mehanna, A. S.; Wireko, F. C.; Whitney, J.;    Thomas, R. P.; Orringer, E. P. Vanillin, a potential agent for the    treatment of sickle cell anemia. Blood 1991, 77, 1334-1341.-   7. Zaugg, R. H.; Walder, J. A.; Walder, R. Y.; Steele, J. M.;    Klotz, I. M. Modification of hemoglobin with analogs of aspirin. J.    Biol. Chem. 1980, 255, 2816-2821.-   8. Abdulmalik, P.; Ghatge, M. S.; Musayev, F. N.; Parikh, A.; Chen,    Q.; Yang, J.; Nnamani, I. N.; Danso-Danquah, R.; Eseonu, D. N.;    Asakura, K.; Abraham, D. J.; Venitz, J.; Safo, M. K.    Crystallographic analysis of human hemoglobin elucidates the    structural basis of the potent and dual antisickling activity of    pyridyl derivatives of vanillin. Acta Crystallogr. D Biol.    Crystallogr. 2011, D67, 920-928.-   9. Rhoda, M. D.; Martin, J.; Blouquit, Y.; Garel, M. C.;    Edelstein, S. J.; Rosa, J. Sickle cell hemoglobin fiber formation    strongly inhibited by the Stanleyville II mutation (alpha 78 asn    leads to lys), Biochem. Biophys. Res. Commun. 1983, 111, 8-13.-   10. Safo, M. K.; Abraham, D. J. X-ray crystallography of    hemoglobins. Methods Mol. Med. 2003, 82, 1-19.-   11. Abdulmalik, O.; Safo, M. K.; Chen, Q.; Yang, J.; Brugnara, C.;    Ohene-Frempong, K.; Abraham, D. J.; and Asakura, T.    5-hydroxymethyl-2-furfural modifies intracellular sickle haemoglobin    and inhibits sickling of red blood cells. Br. J. Haematol. 2005,    128, 552-561.-   12. Xu, G. G.; Pagare, P. P.; Ghatge, M. S.; Safo, R. P.; Gazi, A.;    Chen, Q.; David, T.; Alabbas, A. B.; Musayev, F. N.; Venitz, J.;    Zhang, Y.; Safo, M. K.; Abdulmalik, O. Design, Synthesis, and    Biological Evaluation of Ester and Ether Derivatives of Antisickling    Agent 5-HMF for the Treatment of Sickle Cell Disease. Mol Pharm.    2017, 14, 3499-3511.-   13. Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.;    Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.;    Leslie, A. G. W.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.;    Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin,    A.; Wilson, K. S. Overview of the CCP4 Suite and Current    Developments. Acta Crystallogr. D Biol. Crystallogr. 2011, 67 (Pt    4), 235-242.-   14. Echols, N.; Grosse-Kunstleve, R. W.; Afonine, P. V.; Bunkóczi,    G.; Chen, V. B.; Headd, J. J.; McCoy, A. J.; Moriarty, N. W.;    Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T.    C.; Adams, P. D. Graphical Tools for Macromolecular Crystallography    in PHENIX. J. Appl. Crystallogr. 2012, 45 (Pt 3), 581-586.-   15. Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and    Development of Coot. Acta Crystallogr. D Biol. Crystallogr. 2010, 66    (Pt 4), 486-501.-   16. Brünger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,    P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges,    M.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.;    Warren, G. L. Crystallography & NMR System: A New Software Suite for    Macromolecular Structure Determination. Acta Crystallogr. D Biol.    Crystallogr. 1998, 54 (Pt 5), 905-921.

Example 2 General Procedure to Prepare the Thiozolidine ModifiedCompounds

To a stirring solution of L-cysteine ethyl ester hydrochloride (15.9mmol) and ethyl-diisopropyl amine (15.9 mmol) in anhydrous ethanol (30mL) at room temperature is added dropwise an equimolar amount of thealdehyde compound (15.9 mM) in anhydrous ethanol (20 mL). The reactionmixture is stirred at the same temperature for 2 hrs or overnight. Themixture is then diluted with water (100 mL) and the product extractedwith ethyl acetate (3×50 mL). The organic phase is dried, evaporated,and the product purified through a column with EtOAc:Hexanes as theeluent.

While the invention has been described in terms of its several exemplaryembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1. A vanillin-derived compound having Formula I:

where R1 and R2 are the same or different and are hydrogen, hydroxyl,halogen, or a substituted or unsubstituted alkyl, alkoxy, aryl, O-aryl,cycloalkane or heterocycle; R3 is alkyl ester, aryl ester, alkylamide,arylamide, phosphate or sulfate; M and Q are the same or different andare O or (CH₂)n where n=0-6; X, Y, Z, W and V are the same or differentand are independently H, C, N, S or O; m=0-6, and P=CHO or a promoiety;or a pharmaceutically acceptable salt thereof.
 2. The vanillin-derivedcompound of claim 1, wherein the vanillin-derived compound is designedto bind to the F helix of hemoglobin (Hb).
 3. The vanillin-derivedcompound of claim 1 wherein P is CHO.
 4. The vanillin-derived compoundof claim 1 where P is a protected aldehyde group or promoiety.
 5. Thevanillin-derived compound of claim 4, wherein the promoiety is

where R4 is H or a linear or branched C1-C5 alkyl; and where the bondmarked with * bonds directly to a carbon of the benzene ring.
 6. Thevanillin-derived compound of claim 1, which is selected from the groupconsisting of:


7. The vanillin-derived compound of claim 6, wherein thevanillin-derived compound is


8. The vanillin-derived compound of claim 7, wherein thevanillin-derived compound is


9. The vanillin derived compound of claim 1, wherein thepharmaceutically acceptable salt is an HCl salt.
 10. A compositioncomprising, at least one vanillin-derived compound of claim
 1. 11. Thecomposition of claim 10, which is in a form for oral administration. 12.A method of preventing or treating one or more symptoms or conditions ofsickle cell disease (SCD) in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of atleast one vanillin-derived compound of claim
 1. 13. The method of claim12, wherein the one or more symptoms or conditions of SCD are selectedfrom the group consisting of HbS polymerization, red blood cell (RBC)sickling, adhesion of RBCs to tissue endothelium, oxidative stressand/or damage, hemolysis of RBCs, inflammation, vaso-occlusion, impairedmicrovascular blood flow, a decrease in vascular nitric oxidebioavailability, pain, and death.
 14. The method of claim 12, whereinthe step of administering is performed orally.
 15. A method ofpreventing or treating one or more symptoms of hypoxia in a subject inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of at least one vanillin-derived compound of claim 1.16. The method of claim 15, wherein the step of administering isperformed orally.